1.
 Separation of Proteins in Whey by Strong Anion-Exchange Membrane of Q Sepharose
  

Q Sepharose
      

* ^†

  , *  

(2002 3 20 , 2002 5 30 )

Separation of Proteins in Whey by Strong Anion-Exchange Membrane of Q Sepharose

Du Young Choi* and Kyung Ho Row^†

*Center for Advanced Bioseparation Technology, Dept. of Chem. Eng., Inha University, Inchon 402-751, Korea

(Received 20 March 2002; accepted 30 May 2002)



            α-lactalbumin, BSA, β-lactoglobulin A

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Abstract−Strong anion-exchange membrane in the linear gradient elution method was experimented for the separation of the major whey proteins of α-lactalbumin, BSA, β-lactoglobulin(A, B). This centrifugal UF filters were used for the pretreat- ment of proteins. The membrane with nominal molecular weight limit 30,000 was more effective than that with 10,000. The salt concentrations were changed to determine the mobile phase composition for separating the whey proteins. The mobile phase was composed of buffer A(20 mM piperazine-HCl pH 6.4) and buffer B(buffer A+1 M NaCl). The optimum mobile phase composition was determined as buffer A/buffer B(vol%)=95/5-90/10(0-5 min), 90/10(5-10 min), 90/10-70/30(10-45 min), 70/

30(45-55 min), 70/30-10/90(55-60 min). With this experimental condition of the linear-gradient elution mode, four major whey proteins were satisfactorily separated.

Key words: Whey Proteins, Anion Exchange Membrane, Buffer, Mobile Phase Composition

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†To whom correspondence should be addressed.

E-mail: rowkho@inha.ac.kr

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anion-exchange membrane(buffer A/buffer B(vol%)=95/5-90/

10(0-5 min), 90/10(5-10 min), 90/10-70/30(10-45 min), 70/30(45- 55 min), 70/30-10/90(55-60 min), 20µµµµl)

Fig. 2. Separation of the standard chemicals of the whey proteins by anion-exchange membrane(mobile phase composition same as in Fig. 1, 20µµµµl).

Fig. 3. Separation of the standard chemicals of the whey proteins by anion-exchange membrane(buffer A/buffer B(vol%)=95/5-90/10 (0-5 min), 90/10(5-10 min), 90/10-87/13(10-20 min), 87/13-70/30 (20-40 min), 70/30-10/90(40-45 min), 20µµµµl).

Fig. 4. Separation of the proteins contained in whey by anion-exchange membrane(mobile phase composition same as in Fig. 3, 20µµµµl, molecular weight limit 10,000 centrifugal UF filters retentated).

 40 4 2002 8

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ß NaCl J Fig. 5. Separation of the proteins contained in whey by anion-exchange

membrane(mobile phase composition same as in Fig. 3, 20µµµµl, molecular weight limit 30,000 centrifugal UF filter permeated).

Fig. 6. Separation of the proteins contained in whey by anion-exchange membrane(buffer A/buffer B(vol%)=95/5-90/10(0-5 min), 90/10 (5-10 min), 90/10-70/30(10-45 min), 70/30(45-55 min), 70/30-10/90 (55-60 min), 20µµµµl, no use centrifugal UF filter).

Fig. 7. Separation of the proteins contained in whey by anion-exchange membrane(mobile phase composition same as in Fig. 6, 20µµµµl, molecular weight limit 10,000 centrifugal UF filter retentated).

Fig. 8. Separation of the proteins contained in whey by anion-exchange membrane(mobile phase composition same as in Fig. 6, 20µµµµl, molecular weight limit 30,000 centrifugal UF filter permeated).

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ƒ F* ¹[ æçJMN a£#. U )C $ (o

  Ÿ5N gFPÛª, uvwq[ gÛPŽ Þ9X Cm  Iºª, ŸN «Fb C In 3-P#. ä ™O2I  U ‚l ûdNr¯ α-lactalbumin, BSA  β-lactoglobulin A, β-lactoglobulin B[ ŸP hn tO Ž  dN /)… 4 "

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Fig. 9. Separation of the proteins contained in whey by anion-exchange membrane(mobile phase composition same as in Fig. 6, 50µµµµl, no use centrifugal UF filter).

Fig. 10
Separation of the proteins contained in whey by anion-exchange membrane(mobile phase composition same as in Fig. 6, 50µµµµl, molecular weight limit 10,000 centrifugal UF filter retentated).

Fig. 11. Separation of the proteins contained in whey by anion-exchange membrane(mobile phase composition same as in Fig. 6, 50µµµµl, molecular weight limit 30,000 centrifugal UF filter permeated).

Table 1. Molecular masses and isoelectric points for the whey proteins

Protein Proportion of skim

milk protein(%)

Concentration in whey(g/l)

Proportion of total whey protein(%)

Isoelectricpoint (pI)

Molecular mass

α-lactalbumin 2-5 0.7 12 4.2-4.5 14,000

Bovine serum albumin(BSA) 0.7-1.3 0.3 5 5.13 69,000

β-lactoglobulin 7-12 3.0 50 5.35-5.49 18,300

Immunoglobulins 1.9-3.3 0.6 10 5.5-8.3 15,000-1,000,000

 40 4 2002 8

4. Ye, X., Yoshida, S. and Ng, T. B.: The Int. J. of Biochem. & Cell Biology, 32, 1143(2000).

5. Korhonen, H., Pihlanto-Leppala, A., Rantamaki, P. and Tupasela, T.: Trends in Food Science & Technology, 9, 307(1998).

6. Bury, D., Jelen, P. and Kimura, K.: Dairy Journal, 8, 149(1998).

7. Lieske, B. and Konrad, G.: Dairy Journal, 6, 13(1996).

8. Gerberding, S. J. and Byers, C. H.: J. Chormatogr. A, 808, 141(1998).

9. Splitt, H., Mackenstedt, I. and Freitag, R.: J. chromatogr. A, 729, 87(1996).

10. Zydney, A. L.: Dairy J., 8, 243(1998).

11. Hahn, R., Schulz, P. M., Schaupp, C. and Jungbauer, A.: J. Chor- matogr. A, 795, 277(1998).

20. Josic, D., Buchacher, A. and Junhbauer, A.: J. chromatogr. B, 752, 191 (2001).

21. Splitt, H., Mackenstedt, I. and Freitag, R.: J. Chromatogr. A, 729, 87 (1996).

22. Manji, B., Hill, A., Kakuda, Y. and Irvine, D. M.: J. Dairy Sci., 68, 3176(1985).

23. Shan, L. and Anderson, D. J.: J. Chromatogr. A, 909, 191(2001).

24. Keith Roper, D. and Lightfoot, E. N.: J. Chromatogr. A, 702, 3(1995).

25. Koo, Y. M. and Row, K. H.: J. Korean Ind. Eng. Chem., 11, 807(2000).

26. http://100.empas.com/entry.html/?i=52227&Ad=kfc.